ELISA assay

Microglial cells (6 × 10⁵ cells) were seeded onto a 6-well culture plate, after 24 h cells were stimulated with LPS 100 ng/ml + IFNγ 20 ng/ml or LPS 100 ng/ml + IFNγ 20 ng/ml + CXCL16 (200 nM) for 24 h. Medium was than collected, centrifuged at 1,000 × g for 20 min, and supernatant was stored at −80°C. Control cells were stimulated only with vehicle. IL-1β present in the supernatant was measured using a specific ELISA for mouse IL-1β (Cloud-Clone Corp.) as described by the manufacturer. For each sample, cells were detached and proteins were quantified (BCA assay). For quantification of mouse CXCL16 in glioma conditioned medium (GCM) we used the mouse CXCL16 ELISA Kit (RayBiotech, Norcross, GA, USA) as described by the manufacturer. All supernatants were centrifuged (1,000 × g for 5 min) to eliminate floating cells and then samples were 10-fold concentrated with 10 KDa Microcon Centrifugal Filter devices (Merck Millipore, Darmstadt, Germany). Samples were measured in duplicate and confirmed in two independent experiments.